Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted (99m)Tc-radioimaging agents

Abstract

The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted (99m)Tc-chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 +/- 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMA-targeted chemotherapy.

Figure 1.

(a) Structures of DUPA and GPI-18431. (b–d) Structures were docked into the active site of NAALADase (PSMA) using the computer program GLIDE as described in the Experimental Section. Superimposition of (b) docked DUPA (green) with GPI-18431 (brown) and (c) docking interactions of DUPA (green) with residues in the active site of NAALADase. The protein is depicted in ribbon-cartoon mode, and side chains (indicated in standard one letter code) are depicted in stick mode. Yellow arrow indicates γ′-carboxylic acid of DUPA. The surface representation of the gradually narrowing 20 Å deep tunnel that extends from the protein surface to the active site [(d) top view and (e) side view]. T1, T2, and T3 label the cavities along the tunnel in panel d.

Figure 2.

Structures of PSMA-targeted radiotracers. X = peptide spacer, in which amino acids are indicated using their three letter abbreviations.

Figure 3.

(a) Binding of PSMA-targeted radiotracers containing different spacers to PSMA-positive human prostate cancer cells (LNCaP) in culture. (b) Competitive inhibition of the binding of the 3 best DUPA conjugates (compounds 4, 5, and 6) with excess PMPA is shown in the middle panel. This nearly quantitative competition demonstrates that DUPA conjugate binding is highly specific. (c) Binding of compound 5 to cultured LNCaP cells for 1 h at 4 and 37 °C was compared as a means of assessing the rate of internalization of the DUPA-targeted conjugate. Error bars represent SD (n = 3).

Figure 4.

(a) Molecular dynamics prediction of the superimposition of radioimaging agent 5 (cyan) with DUPA (green) and GPI-18314 (brown) in the active site of the protein. The protein is depicted in ribbon-cartoon mode, and side chains are depicted in stick mode (indicated in standard one letter code). Binding modes of compounds 5 [(b), (c) side and (f) top view (hydrophobic sites = green, hydrophilic sites = blue, surfaces exposed to solvent = red)], (d) compound 3 and (e) compound 6.

Figure 5.

Overlay of whole-body radioimages (rainbow colors) on white light photographs of mice bearing LNCaP tumor xenografts that were treated with radioimaging agents (a) 3, (b) 4, (c) 5, (d) 5 (competition with 100-fold excess PMPA), (e) 6, and (f) 7. Kidneys were shielded with a lead plate.

Figure 6.

Biodistribution of radioimaging agents in nu/nu mice bearing LNCaP tumor xenografts. Error bars represent SD (n = 4).

Scheme 1

a ^a Reagents and conditions: (a) triphosgene, TEA/ DCM, −78 °C; (b) H-l-Glu(OBn)-O^tBu • HCl; (c) H₂; Pd–C/DCM.

Scheme 2

a ^a Reagents and conditions: (a) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-Asp(O^tBu)-OH, HBTU, HOBt, DIPEA, 2 h; (b) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-diaminopropionic (DAP) acid, HBTU, HOBt, DIPEA, 2 h; (c) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-Phe-OH, HBTU, HOBt, DIPEA, 2 h; (d) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-Phe-OH, HBTU, HOBt, DIPEA, 2 h; (e) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-8-aminooctanoic (EAO) acid, HBTU, HOBt, DIPEA, 2 h; (f) (i) 20% piperidine/DMF, rt, 10 min; (ii) 9, HBTU, HOBt, DIPEA, 2 h; (g) TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5), 1 h; (h) SnCl₂, sodium glucoheptonate/H₂O; (i) aq NaHCO₃/pH = 6.8–7.2; (j) sodium pertechnetate/saline, 100 °C, 18 min.